Project Summary/Abstract Hepatocellular carcinoma (HCC) is the most common primary liver cancer and arises from chronic liver diseases (CLD) including hepatitis B virus (HBV), hepatitis C virus (HCV), alcoholic liver disease (ALD), and nonalcoholic steatohepatitis (NASH). Today, patients with CLDs are at high risk for HCC and are monitored closely by imaging and serum biomarkers, but these platforms lack sensitivity for early stage HCC. Consequently, although HCC can be cured by surgical resection, less than 10% of the greater than 500,000 newly diagnosed HCC patients per year worldwide undergo surgical resection because of late diagnosis. This proposal aims to develop the Glympse liver test (GLT), a multiplexed, injectable diagnostic of activity-based probes, which detect the activity of proteases that drive the pathological hallmarks of early stage HCC. These HCC-specific proteases are upregulated regardless of etiology and their activities drive fundamental tumor processes including angiogenesis, inflammation, and ECM degradation. The GLT platform is ultrasensitive to early stage HCC by relying on two mechanisms for signal amplification; detection signals are generated by protease activity which amplifies signals by enzyme turnover, and are concentrated by renal filtration into urine for quantification by mass spectrometry. We will identify etiology independent HCC proteases and formulate GLT nanosensors on inert polymer scaffolds for human use. We will then validate the ability of GLT to detect early stage HCC in mouse models covering major etiologies, benchmarking against imaging and serum biomarkers, and then conduct preclinical toxicology studies in preparation for an IND submission. The GLT offers clinician a rapid and sensitive diagnosis of early stage HCC compared to imaging and blood biomarkers. The ability of GLT to detect HCC at an early stage will significantly improve response rates (60?70% at Stage 1), saving 20,000 lives from 30,000 patient deaths every year. The benefit to the healthcare system will be greater than $400 million based on increasing the number of curative resection surgeries while significantly reducing the number of liver transplants. The impact of the GLT for HCC will increase early stage diagnosis rates, reduce healthcare costs associated with treating late-stage HCC, and ultimately improve patient outcomes due to increased treatment options for physicians.